U.S. patent application number 13/357948 was filed with the patent office on 2012-05-17 for undifferentiated stem cell culture systems.
This patent application is currently assigned to Hadasit Medical Research Services & Development Limited. Invention is credited to Benjamin Reubinoff, Debora Steiner.
Application Number | 20120122209 13/357948 |
Document ID | / |
Family ID | 39870128 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120122209 |
Kind Code |
A1 |
Reubinoff; Benjamin ; et
al. |
May 17, 2012 |
Undifferentiated Stem Cell Culture Systems
Abstract
The present disclosure provides methods for maintaining and
propagating undifferentiated pluripotent stem cells (SC) in
suspension. The methods comprise culturing such SC in a
non-adherent culture dish under conditions comprising a basic serum
free medium and one or more of a basic medium, a serum replacement,
an extra cellular matrix component and a factor supporting
expansion of said SC. A specific and preferred culture condition
comprise supplementing Neurobasal.TM. medium with KO serum
replacement (KOSR). These conditions allowed for large scale and
long term propagation of undifferentiated pluripotent SC. The
culture system comprising suspended undifferentiated pluripotent SC
were found to have many applications including in methods for
directed as well as spontaneous differentiation of the SC into
somatic cells. Also disclosed herein is a method of deriving SC,
preferably human embryonic SC from human embryos via the formation
of cell clusters.
Inventors: |
Reubinoff; Benjamin; (Doar
Na Haela, IL) ; Steiner; Debora; (Modi'in,
IL) |
Assignee: |
Hadasit Medical Research Services
& Development Limited
Jerusalem
IL
|
Family ID: |
39870128 |
Appl. No.: |
13/357948 |
Filed: |
January 25, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12570457 |
Sep 30, 2009 |
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13357948 |
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PCT/IL2008/000460 |
Apr 2, 2008 |
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12570457 |
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11730560 |
Apr 2, 2007 |
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PCT/IL2008/000460 |
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Current U.S.
Class: |
435/366 ;
435/375; 435/377; 435/383; 435/384 |
Current CPC
Class: |
C12N 5/0606 20130101;
C12N 2501/115 20130101; C12N 2501/13 20130101; C12N 2501/155
20130101; C12N 2502/1323 20130101; C12N 2501/119 20130101; C12N
2501/16 20130101; C12N 2533/52 20130101; C12N 2533/54 20130101 |
Class at
Publication: |
435/366 ;
435/375; 435/383; 435/384; 435/377 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C12N 5/07 20100101 C12N005/07 |
Claims
1-38. (canceled)
39. A method of expanding undifferentiated pluripotent stem cells
(SC) in suspension, the method comprises: preparing a culture
system comprising a non adherent culture dish, a basic medium; a
serum replacement, a factor that promote maintenance of
pluripotency, an extracellular matrix component and suspended
therein said SC; and allowing said SC to expand; whereby the
expanded SC are maintained in an undifferentiated, pluripotent
state.
40. The method of claim 39, wherein said culture system comprises a
basic medium; a Knockout.TM. serum replacement (KOSR) and
undifferentiated SC suspended therein and the method comprises
incubating said SC in said culture system for a period of time
allowing said cells to expand, the expanded cells being in an
undifferentiated pluripotent state.
41. The method of claim 40, wherein said undifferentiated SCs are
derived from SC colonies cultivated on a feeder layer or in a
feeder free adherent culture system.
42. The method of claim 40, wherein the undifferentiated SCs in
said suspension are in form of free floating cells, free floating
clusters of cells or free floating aggregates of cells.
43. The method of claim 40, wherein said basic medium is
Neurobasal.TM..
44. The method of claim 40, wherein said culture system comprises
one or more of the following: a member of FGF family; a
extracellular matrix (ECM) component; an antibacterial agent;
nonessential amino acids; a TGF.beta. superfamily factor; a
neurotrophin; nicotinamide (NA); a bone morphogenic protein (BMP)
antagonist; or a serum free medium supplement.
45. The method of claim 44, wherein said ECM component is selected
from fibronectin, laminin and gelatin; said antibacterial agent is
selected from penicillin and streptomycin; said TGF.beta.
superfamily factor is selected from activin A; said BMP antagonist
is selected from noggin, chordin; and said serum free medium
supplement is selected from Nutridoma-CS or TCH.TM..
46. The method of claim 39, for one or more of large-scale
expansion and long term cultivation of undifferentiated pluripotent
SCs in suspension.
47. A culture system for expansion of stem cells (SCs) comprising a
suspension of undifferentiated stem cells within a basic medium and
KOSR, wherein said stem cells are selected from the group
consisting of free floating cells, free floating clusters of cells
and free floating aggregates of cells.
48. The culture system of claim 47, wherein said the basic medium
is Neurobasal.TM..
49. The culture system of claim 47, comprising one or more of the
following: a member of FGF family; an extracellular matrix (ECM)
component; an antibacterial agent; non-essential amino acids a
TGF.beta. superfamily factor; a neurotrophin; nicotinamide (NA); a
bone morphogenic protein (BMP) antagonist; or a serum free medium
supplement.
50. The culture system of claim 47, wherein said FGF member is
FGF-2, ECM component is selected from fibronectin, laminin and
gelatin; said antibacterial agent is selected from penicillin and
streptomycin; said TGF.beta. superfamily factor is activin A; said
BMP antagonist is selected from noggin, chordin, or gremlin, said
neurotrophin is selected from BDNF, NT3, NT4; and said a serum free
medium supplement is selected from Nutridoma-CS or TCH.TM..
51. The culture system of claim 47, wherein said SCs are
pluripotent, human embryonic SCs (hESCs).
52. A method for directing differentiation of SCs into a selected
population of somatic cells from a culture system of SCs in
suspension, the method comprising: (a) providing a suspension of
undifferentiated SCs obtained by the method of claim 39, wherein
the culture system comprises a basic medium and Knockout.TM. serum
replacement (KOSR); and (b) incubating said undifferentiated SCs in
a culture system that supports directed differentiation of SCs into
the selected fate of somatic cells.
53. The method of claim 52, wherein said SCs are hESC.
54. The method of claim 52, wherein the SCs are incubated in a
culture system that directs differentiation into somatic cells are
selected from mesodermal, endodermal or ectodermal cells.
55. The method of claim 54, wherein said selected population of
somatic cells consists essentially of a single population of
differentiated somatic cells.
56. The method of claim 54, wherein said population of somatic
cells consists essentially of neural precursor or neural stem
cells.
57. The method of claim 54, wherein the culture system directing
differentiation comprises a basic media, FGF2 and/or noggin.
58. The method of claim 57, wherein said selected population of
somatic cells consists essentially of dopaminergic neuronal
cells
59. The method of claim 58, wherein said culture system supporting
differentiation of neural precursor cells into dopaminergic
neuronal cells comprises at least one of sonic hedgehog (SHH), a
fibroblast growth factor (FGF), or a member of the Wnt family
followed by final differentiation in the presence of at least one
of FGF, member of the Wnt family and survival factors.
60. A method for promoting spontaneous differentiation of SCs into
somatic cells, the method comprising: (a) providing a suspension of
undifferentiated pluripotent SCs obtained by the method of claim 39
wherein the culture system comprises a basic medium and
Knockout.TM. serum replacement (KOSR); (b) incubating said
undifferentiated SCs in culture system that support spontaneous
differentiation of SCs into the somatic cells.
61. The method of claim 60, wherein said somatic cells comprise a
mixture or any one of mesodermal cells, ectodermal cells,
endodermal cells and combination of same.
62. The method of claim 60, wherein said culture system that
supports spontaneous differentiation of SCs into somatic cells
comprises a basic medium of DMEM supplemented by FCS 20%.
63. A method for deriving cells from a human embryo the cells
exhibiting a morphological characteristic of a human embryonic stem
cell, the method comprising: (a) providing in vitro fertilized
embryos; (b) culturing said embryos to a blastocyte stage; (c)
isolating from said blastocyte inner cell mass (ICM); (d) culturing
said ICM in suspension with a feeder free culture system comprising
a basic media and a serum replacement until clusters capable of
propagating are formed; wherein said clusters comprise cells
exhibiting at least one morphological characteristic of a SC.
Description
FIELD OF THE INVENTION
[0001] This invention relates to stem cells (SC), and particularly
to methods and systems for handling and propagating human embryonic
stem cells (hESC).
LIST OF RELATED ART
[0002] The following is a list of references which are considered
to be pertinent for describing the state of the art in the field of
the invention. [0003] (1) Reubinoff, B. E., Pera, M. F., Fong, C.
Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from
human blastocysts: somatic differentiation in vitro. Nat Biotechnol
18, 399-404 (2000) [0004] (2) Amit, M. et al. Clonally derived
human embryonic stem cell lines maintain pluripotency and
proliferative potential for prolonged periods of culture. Dev Biol
227, 271-278 (2000). [0005] (3) Xu, C. et al. Feeder-free growth of
undifferentiated human embryonic stem cells. Nat Biotechnol 19,
971-974 (2001). [0006] (4) Amit, M. et al. Human feeder layers for
human embryonic stem cells. Biol Reprod 68, 2150-2156 (2003).
[0007] (5) Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C.
& Bongso, A. Human feeders support prolonged undifferentiated
growth of human inner cell masses and embryonic stem cells. Nat
Biotechnol 20, 933-936 (2002). [0008] (6) Cowan, C. A. et al.
Derivation of embryonic stem-cell lines from human blastocysts. N
Engl J Med 350, 1353-1356 (2004). [0009] (7) Amit, M., Shariki, C.,
Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and
serum-free culture of human embryonic stem cells. Biol Reprod
70(3):837-45 (2004). [0010] (8) Pera, M. F. et al. Regulation of
human embryonic stem cell differentiation by BMP-2 and its
antagonist noggin. J Cell Sci 117, 1269-1280 (2004). [0011] (9) GB
2409208. [0012] (10) WO 04/031343. [0013] (11) Xu, R. H., et al.
Basic FGF and suppression of BMP signaling sustain undifferentiated
proliferation of human ES cells. Nat Methods. 3, 164-5 (2005).
[0014] (12) Vallier L, et al. Activin/Nodal and FGF pathways
cooperate to maintain pluripotency of human embryonic stem cells. J
Cell Sci. 118, 4495-509 (2005). [0015] (13) WO 06/070370
(Reubinoff, B. et al.). [0016] (14) Itsykson, P., et al. Derivation
of neural precursors from human embryonic stem cells in the
presence of noggin. Mol Cell Neurosci 30(1), 24-36 (2005). [0017]
(15) Yan, Y. et al. Directed differentiation of dopaminergic
neuronal subtypes from human embryonic stem cells. Stem Cells
23(6), 781-90 (2005). [0018] (16) Ludwig, T. E. et al. Derivation
of human embryonic stem cells in defined conditions. Nat Biotechnol
24(2), 185-87 (2006). [0019] (17) Kallos, M. S. et al. Large-scale
expansion of mammalian neural stem cells: A review. Med Biol Eng
Comput 41(3), 271-82 (2003). [0020] (18) Cormier, J. T., et al.
Expansion of Undifferentiated Murine Embryonic Stem Cells as
Aggregates in Suspension Culture Bioreactors. Tissue Eng 1, 1
(2006). [0021] (19) Fok, E. Y. et al. Shear-controlled single-step
mouse embryonic stem cell expansion and embryoid body-based
differentiation. Stem Cells 23(9), 1333-42 (2005). [0022] (20)
Gerecht-Nir, S. et al. Bioreactor cultivation enhances the
efficiency of human embryoid body (hEB) formation and
differentiation. Biotechnol Bioeng 86(5), 493-502 (2004). [0023]
(21) Goldsborough, M. D. et al. Serum-free culture of murine
embryonic stem (ES) cells. Focus 20(1), 8-12 (1998). [0024] (22) WO
98/30679. [0025] (23) WO 03/104444. [0026] (24) Reubinoff B E,
Khaner H. Identification and Maintenance of Neural Progenitors from
human ES cells. In Handbook Of Stem Cells, Volume 2: Embryonic Stem
Cells. Lanza R, Gearhart J D, Hogan B L M, Mckay R D, Melton D A,
Pedersen R, Thomson J A West M D (eds). Academic Press 2004, pages
511-520. [0027] (25) Klimanskaya I, Chung Y, Becker S, Lu S J,
Lanza R. Human embryonic stem cell lines derived from single
blastomeres. Nature. 2007 Mar. 15; 446(7133):342. [0028] (26)
D'Amour K A, Bang A G, Eliazer S, Kelly O G, Agulnick A D, Smart N
G, Moorman M A, Kroon E, Carpenter M K, Baetge E E. Production of
pancreatic hormone-expressing endocrine cells from human embryonic
stem cells. Nat Biotechnol. 2006 November; 24(11):1392-401. [0029]
(27) Yao S, Chen S, Clark J, Hao E. Beattie G M Hayek A and Ding S.
Long-term self-renewal and directed differentiation of human
embryonic stem cells in chemically defined conditions. PNAS 2006;
103: 6907-6912.
BACKGROUND OF THE INVENTION
[0030] Stem cells are immature, unspecialized cells that renew
themselves for long periods through cell division. Under certain
conditions, they can differentiate into mature, functional cells.
Human embryonic stem cells (hESC) are derived from early surplus
human blastocysts'. Human ES cells are unique stem cells since they
can self-renew infinitely in culture, and since they have a
remarkable potential to develop into extraembryonic lineages as
well as all somatic cells and tissues of the human body.sup.1.
[0031] Given the unique properties of hESC, they are expected to
have far-reaching applications in the areas of basic scientific
research, pharmacology, and regenerative medicine. Human ES cell
lines can provide a powerful in vitro model for the study of the
molecular and cellular biology of early human development, for
functional genomics, drug screening, and discovery. They may serve
for toxicology and teratogenicity high throughput screening. Since
hESC can self-renew indefinitely and can differentiate into any
cell type, they can serve as a renewable, unlimited donor source of
functionally mature differentiated cells or tissues for
transplantation therapy. In addition, transplanted
genetically-modified hESC can serve as vectors to carry and express
genes in target organs in the course of gene therapy.
[0032] While the promise of hESC for basic scientific research
pharmacology and regenerative medicine is remarkable, the
exploitation of hESC for most applications depends upon further
development. Improved control of the growth of undifferentiated
hESC, the development of bulk feeder-free cultures of
undifferentiated cells, the development of animal-free culture
systems, and the development of methods and tools which direct the
differentiation and generate pure cultures of mature functional
cells of a specific type are required.
[0033] At present, a few culture systems are most commonly used to
propagate undifferentiated hESC.sup.1-3. In the initial culture
system that was developed, undifferentiated hESC are cultured in
serum-containing medium as colonies, upon a layer of fibroblast
feeder cells (of mouse.sup.1 or human origin.sup.4, 10) It is
possible to remove all animal products from this culture system and
replace them with those from a human source.sup.5. It was found
that in this system the cells are propagated as clumps on a small
scale, which does not allow efficient cloning.sup.2.
[0034] An alternative culture system for use in the proliferation
of undifferentiated growth of hESC comprises a culture matrix
comprising extracellular matrix (ECM) that may be prepared from
feeder cells or other sources and a conditioned medium being
preconditioned by feeder cells. The suggested leading cells in the
feeder cells include primary mouse embryonic fibroblasts (PMEF), a
mouse embryonic fibroblast cell line (MEF), murine foetal
fibroblasts (MFF), human embryonic fibroblasts (HEF), human foetal
muscle (HFM), human foetal skin cells (HFS), human adult skin
cells, human foreskin fibroblasts (HFF).sup.9, human adult
Fallopian tubal epithelial cells (HAFT), or human marrow stromal
cells (HMSC).
[0035] Another alternative culture system that was developed and
used extensively is a serum-free system that includes the knockout
(KO) medium supplemented with knockout serum replacement (KOSR) and
FGF2. This system allows cloning of undifferentiated hESC, although
at a low efficiency.sup.2. Undifferentiated cells are cultured as
flat colonies and may be propagated mechanically as small clusters
or single cells (by using trypsin.sup.6).
[0036] Knockout serum replacement (KOSR) (Gibco) is a chemically
defined, serum-free culture medium supplement used as a substitute
for animal-based serum in KO-DMEM-based culture systems (KO-DMEM
e.g. of catalog no. 10829-018 Gibco 1998/1999) for propagating stem
cells. KOSR can efficiently promote the growth and maintenance of
undifferentiated embryonic stem cells and therefore may replace the
supplementation with fetal bovine serum (FBS).sup.(21). KO-DMEM may
replace traditional DMEM in either FBS- or KOSR-supplemented
cultures.sup.(21).
[0037] Undifferentiated propagation of adherent colonies of hESCs
may be accomplished with a KO serum-free culture system without the
use of feeders by plating and growing the colonies on extracellular
matrices (ECM) within a feeder-conditioned KO-DMEM medium
supplemented with KOSR and FGF2.sup.3. Furthermore, it has been
suggested that feeder conditioning may be replaced by substituting
the medium with high concentrations of FGF2 and noggin.sup.11.
Alternatively, feeder conditioning was replaced by transforming
growth factor .beta.1 and human LIF (in addition to FGF2) and to
growing the cells on human fibronectin.sup.7, or by serum-free
media supplemented with soluble factors including FGF2, activin A,
transforming growth factor .beta.1 (TGF.beta.1), pipecolic acid,
GABA, LiCL and culturing the cells on ECM components (16). In
another recent publication, undifferentiated propagation of hESC
colonies, in the absence of feeders, was reported with a chemically
defined medium without serum replacer, supplemented with activin or
nodal plus FGF2.sup.12. In general, a key limitation of hESC
culture systems is that they do not allow the propagation of pure
populations of undifferentiated stem cells and their use typically
involves some level of background differentiation. The stem cells
most commonly follow a default pathway of differentiation into an
epithelial cell type that grows either as a monolayer of flat
squamous cells or form cystic structures. Most probably, this form
of differentiation represents differentiation of hESC into
extraembryonic endoderm.sup.8.
[0038] In these adherent culture systems of colonies, the hESCs are
most commonly passaged (mechanically and/or by using enzymatic
digestion) and re-plated as clusters, on a small scale. These
culture systems are labor-intensive, highly variable, may contain
undefined factors, and do not provide steady-state operating
conditions. Most importantly, they do not typically allow for large
scale production of standardized homogenous undifferentiated hESCs
needed for the aforementioned uses.
[0039] Suspension culture bioreactors offer several advantages over
the conventional use of static monolayer cultures. These systems
facilitate the large-scale expansion of the cells in a homogeneous
culture environment, thus decreasing the risk of culture
variability. They are also less labor-intensive to operate and
offer the possibility of computer control and monitoring of the
culture conditions. Although bioreactors have been used to expand
neural stem cells.sup.(17), mouse ES cells.sup.(18) and
differentiating hESCs within embryoid bodies (EBs)(20), only
recently some progress has been made towards the development of
protocols for the feeder-free expansion of undifferentiated hESCs
in suspension systems.sup.13.
[0040] A major obstacle in developing systems for culturing hESCs
in suspension bioreactors was recently overcome. Until recently it
was only possible to grow undifferentiated hESCs as flat cultures.
Culturing clusters of hESCs in suspension induces their
differentiation within EBs and is most commonly used to induce
differentiation. Surprisingly, and against the accepted concept
that hESCs in suspension undergo differentiation, it was recently
demonstrated that hESCs may be propagated, in the pluripotent,
undifferentiated state, as clusters in suspension using
Neurobasal.TM. medium as the basic medium of the culture
system.sup.(13), which is supplemented with N2. Neurobasal.TM.
medium is a basal medium especially formulated for growth of
neuronal cells, and supplemented with either serum (e.g., FBS) or
B27 or N2 serum replacement.sup.23.
SUMMARY OF THE INVENTION
[0041] It has been envisages that when providing a culture system
where SC are cultured in a non-adherent culture dish, with one or
more of a basic serum free medium, a serum replacement, an extra
cellular matrix component and a factor supporting expansion of said
SC, such conditions allow maintenance and expansion of the SC in an
undifferentiated pluripotent state.
[0042] The present disclosure is based on the finding that
supplementing Neurobasal.TM. (Catalog no. 21103-049, Gibco
1998/1999) medium with KO serum replacement (KOSR, Catalog No.
10828 Gibco 1998/1999) allowed for the significant expansion of
undifferentiated and pluripotent stem cells cultivated in a
suspension. Previously, it was shown that undifferentiated and
pluripotent stem cells cultivated as suspended cells (i.e. in a
suspension) may be expanded in a culture medium supplemented with
N2 (WO 2006/070370).
[0043] In a specific preferred aspect it has been surprisingly
found that when using KOSR (instead of with N2) a significant
increased level of expansion of undifferentiated and pluripotent
stem cells cultivated in a suspension is exhibited (at least double
of that obtained with N2). These findings were unexpected as KOSR
is typically used in the art for inducing differentiation of stem
cells in clusters.
[0044] Specifically, the results provided hereinbelow show that
after 3 weeks the total number of undifferentiated and pluripotent
human embryonic stem cells (hESCs) propagated in
Neurobasal.TM.-KOSR (NBSR) medium reached a population of
approximately double that of Neurobasal.TM.-N2 (NBN2)-cultured
hESCs.
[0045] Further, it was surprisingly found that the NBSR medium
could effectively support long-term cultivation of undifferentiated
pluripotent hESCs. The specific, non-limiting examples provided
hereinbelow show that the percentage of hESCs expressing markers of
pluripotent stem cells (SSEA4, TRA1-60, and TRA1-81) was high
(>95%) and stable after 3.5 weeks, 6.5 weeks as well as after 20
weeks of suspension culture.
[0046] Furthermore, it was surprisingly found that NBSR medium
supplemented with Nutridoma-CS (Catalog no. 11363743001 Roche 2006)
did not require the addition of laminin for culturing
undifferentiated hESCs in suspension for up to 6 weeks and only
minimal concentrations of laminin (at most 10 ng/ml) for longer
culture periods (above 6 weeks and even up to 20 weeks).
[0047] Thus, in accordance with its broadest aspect, there is
provided by the present disclosure a method of expanding in
suspension SCs in an undifferentiated pluripotent state, the method
comprises preparing a culture system comprising a basic medium; a
serum replacement, factors that promote the maintenance of
pluripotency, non adherent culture dishes, and extracellular matrix
component, the combination of which supports expansion of SC in an
undifferentiated state, and suspended therein, undifferentiated SC;
and incubating said SC for a period of time allowing the cells to
expand, the expanded cells being maintained in an undifferentiated
pluripotent state.
[0048] A preferred embodiment of this aspect concerns a method of
expanding in suspension SCs in an undifferentiated pluripotent
state, the method comprises preparing a culture system comprising
one or more of a basic medium; and knock out serum replacement
(KOSR) or an acceptable variation or alternative thereof, and
suspended therein, undifferentiated pluripotent SC; and incubating
the SC for a period of time allowing the cells to expand, the
expanded cells being maintained in said pluripotent
undifferentiated state.
[0049] Further in accordance with its broadest aspect, there is
disclosed herein, a culture system comprising a suspension of SC in
an undifferentiated pluripotent state suspended in a basic medium
supplemented with one or more of a serum replacement, a factor that
supports maintenance of pluripotency, a non adherent culture dish,
and an extracellular matrix component, the combination elements
supporting expansion of said SC in an undifferentiated pluripotent
state.
[0050] A preferred embodiment of this aspect concerns a culture
system comprising stem cells in an undifferentiated pluripotent
state suspended in a basic medium supplemented with KOSR, the
culture system allowing expansion of said cells while maintaining
their undifferentiated, pluripotent state.
[0051] In accordance with a preferred embodiment of the aspects
disclosed hereinabove, the serum replacement is knockout serum
replacement (KOSR). In accordance with another preferred embodiment
of this aspect, the basic medium is Neurobasal.TM..
[0052] In accordance with a third aspect, there is provided the use
of KOSR (or an acceptable alternative thereof) in combination with
a basic medium for the preparation of a culture system for
maintaining in suspension undifferentiated pluripotent SCs.
[0053] In accordance with a fourth aspect, there is provided a use
of the suspension of the undifferentiated pluripotent SC obtainable
by the method disclosed herein in a method of directing
differentiation of SCs into a selected population of somatic cells
from a culture system of SCs in suspension, the method comprising:
[0054] (a) providing a suspension of undifferentiated SCs
obtainable by the method disclosed herein; and [0055] (b)
incubating the undifferentiated pluripotent SCs in a culture system
that supports directed differentiation of SCs into a selected fate
of somatic cells.
[0056] In accordance with one embodiment, the selected population
of somatic cells consists essentially of neural precursor cells or
neural stem cells. In accordance with another embodiment the
selected population of somatic cells consists essentially of
neuronal cells including, but not limited thereto, dopaminergic,
gabaergic or glutamatergic cells. Culture systems which support
directed differentiation of SC's into a specifically desirable type
of somatic cell, such as neural precursor cells or dopaminergic
neuronal cells are well known in the art.
[0057] In connection with above directed differentiation method of
SC, there is also provided the use of undifferentiated SC
expandable in suspension obtainable by the method disclosed herein
for the preparation of a composition comprising a selected
SC-derived population of somatic cells.
[0058] In accordance with a fifth aspect, there is provides a use
of the suspension of the undifferentiated pluripotent SC disclosed
herein in a method of promoting spontaneous differentiation of SCs
into somatic cells, the method comprising: [0059] (a) providing a
suspension of undifferentiated SCs obtainable by the method
disclosed herein; [0060] (b) incubating said undifferentiated SCs
in culture system that support spontaneous differentiation of SCs
into the somatic cells.
[0061] The somatic cells in accordance with this aspect may
comprise cells from the three embryonic germ layers: ectoderm;
mesoderm; and endoderm. Thus, the method of the invention for
promoting spontaneous differentiation of somatic cells so as to
provide a population of somatic cells comprising a single cell type
as well as a mixture of ectodermal, mesodermal and/or endodermal
cells.
[0062] Culture systems which support spontaneous differentiation of
SC's into somatic cells are well known in the art. It is noted that
for directed as well as spontaneous differentiation, a preferred
basic medium is KO DMEM.
[0063] Finally, in accordance with a six aspect, there is disclosed
a method for deriving stem cells from human embryos in suspension
the method comprising: [0064] providing in vitro fertilized
embryos; [0065] culturing said embryos to a blastocyte stage;
[0066] isolating from said blastocyte inner cell mass (ICM); [0067]
culturing said ICM in suspension within a feeder free culture
system comprising a non-adherent culture dish, a basic media, serum
replacement, at least one soluble factor that promote the
maintenance of pluripotent stem cells and at least one extra
cellular matrix component until clusters are formed capable of
propagating; [0068] wherein said clusters and cells within them
exhibit morphological characteristics of clusters of
undifferentiated hESC when cultured in suspension.
[0069] Morphological characteristics of clusters of
undifferentiated hESC when cultured in suspension may be recognized
as compacted clusters of uniformly small packed relatively
transparent cells which do not form cystic structures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] In order to understand the invention and to show how it may
be carried out in practice, a preferred embodiment will now be
described, by way of non-limiting example only, with reference to
the accompanying drawings, in which:
[0071] FIG. 1: is a bar graph showing the number of stem cells in a
suspension culture system comprising Neurobasal.TM. (NB) medium as
the basic medium supplemented with KOSR (NBSR) or with N2 (NBN2);
Cell counts were performed 3 weeks after transfer of equal number
of clusters of undifferentiated hESCs into suspension culture
within the two media compositions.
[0072] FIG. 2: is a dark field micrograph of undifferentiated hESCs
in a suspension cultures in NB medium supplemented with KOSR;
[0073] FIGS. 3A-3E: are FACS analysis images of hESC clusters that
were cultured 3 weeks in suspension and dissociated into single
cells and showing that >90% of the cells expressed markers of
pluripotency SSEA-4 (FIG. 3A), TRA1-60 (FIG. 3B), TRA1-81 (FIG. 3C)
and, but not a marker of early neural differentiation, PSA-NCAM
(FIG. 3D). Summary of two independent experiments are presented in
the bar graph histograms (FIG. 3E);
[0074] FIG. 4A-4B are phase contrast image (FIG. 4A) and
fluorescence image (FIG. 4B) of colonies of undifferentiated hESCs
developed after plating clusters that were cultivated in suspension
and then replated on feeders with typical morphology (FIG. 4A), and
with cells within the adherent colonies expressing alkaline
phosphatase (FIG. 4B).
[0075] FIG. 5: is a bar graph showing that supplementation of NB
with Nutridoma-CS, FGF2, activin A, ECM components and neutrophins
promote long term propagation of undifferentiated hESC clusters in
suspension, as exhibited in corresponding FACS analysis that showed
that the percentage of hESCs expressing SSEA-4, TRA1-60, and
TRA1-81 was high (>95%) and stable after 3.5 and 6.5 weeks of
suspension culture.
[0076] FIG. 6: is a bar graph showing that supplementation of NB
Nutridoma-CS however, without laminin also promotes long term
propagation of undifferentiated hESC clusters in suspension, as
exhibited by the expression of the different markers (% positive
cells) in the presence or absence of laminin (+LAM and -LAM,
respectively) and after 3.5 and 6.5 weeks of suspension
culture.
[0077] FIG. 7: is a bar graph showing the number of cells after
long term suspension culture in NBSR supplemented with Nutridoma-CS
being increased after 6.5 weeks in the absence (-LAM) as compared
to the presence (+LAM) of laminin supplementation.
[0078] FIGS. 8A-8C: are phase contrast image (FIG. 8A), indirect
immuno-fluorescence staining image (FIG. 8B) and image of nuclei
counterstaining with DAPI (FIG. 8C) showing cells with
morphological characteristics of neurons emanating from the
clusters after induction in DMEM/F12 supplemented with B27, noggin
and FGF2 for 2-3 weeks and culturing for a week on laminin coated
slides.
[0079] FIG. 9: is an immuno-fluorescent image showing that hESCs
that were propagated in suspension could give rise to midbrain
dopaminergic neurons co-expressing TH (light gray) and EN-1 (dark
gray); The arrow shows a cell which co-expresses EN-1 and TH, after
plating the hESCs on laminin coated slides and culturing in the
presence of FGF8 and SHH for 2 weeks.
[0080] FIG. 10: is an immuno-fluorescent image showing that hESCs
that were propagated in suspension could give rise to mesodermal
cells expressing human muscle actin (hMA).
[0081] FIG. 11: is an immunofluorescent image showing that hESCs
that were propagated in suspension could give rise to endodermal
cells expressing Sox17.
[0082] FIG. 12: is a bar graph showing that NB with Nutridoma-CS,
FGF2, activin A, ECM components and neutrophins promotes long-term
propagation of clusters of undifferentiated hESCs in suspension, as
exhibited in corresponding FACS analysis, which showed that the
percentage of hESCs expressing TRA1-60 and TRA1-81 was high and
stable after 7 and 10 weeks of suspension culture.
[0083] FIGS. 13A-13C: are images of histological sections of
teratoma tumors that developed after inoculation of hESCs,
cultivated in suspension for 7 weeks, into the testes of NOD/SCID
mice. Differentiated progeny representing the three embryonic germ
layers, mesoderm (FIG. 13A), ectoderm (FIG. 13B) and endoderm (FIG.
13C) are illustrated.
[0084] FIG. 14: is a dark field micrograph of undifferentiated
hESCs in a suspension after 7 weeks cultures in NB medium
supplemented with KOSR.
[0085] FIG. 15 is a bar graph histograms of four independent
experiments showing a FACS analysis of hESC clusters that were
cultured 7 weeks in suspension in the presence of Nutridoma.TM.
supplement and dissociated into single cells showing that >90%
of the cells expressed markers of pluripotency SSEA-4, TRA1-60,
TRA1-81 but not a marker of early neural differentiation,
PSA-NCAM;
[0086] FIG. 16 is RT-PCR analysis showing that hESC clusters that
were cultured 7 weeks in suspension express markers of pluripotency
OCT-4, Nanog, Rex-1, and TERT, but not a marker of early
differentiation FGF-5.
[0087] FIGS. 17A-17E are indirect immuno-fluorescence staining
images and images of nuclei counterstained with DAPI (FIGS.
17A-17B, 17D-17E) showing cells expressing markers of neural
precursors (FIG. 17A; nestin, Pax6), neural stem/radial glia cells
(FIG. 17B; 3CB2) and subtypes of neurons including dopaminergig
(FIG. 17C: .beta.-III tubulin and thyrosine hydroxilase (TH)),
gabaergic (FIG. 17D: GABA) and glutamaergic (FIG. 17E: glutmate)
after induction in DMEM/F12 supplemented with B27, noggin and FGF2
for 4 weeks and culturing for a week on laminin coated slides.
[0088] FIG. 18 is a bar graph histograms showing a FACS analysis of
hESC clusters that were cultured 20 weeks in suspension and
dissociated into single cells showing that >95% of the cells
expressed the markers of pluripotency SSEA-4, SSEA-3, TRA1-60 and
TRA1-81 but not the marker of early neural differentiation,
PSA-NCAM;
[0089] FIGS. 19A-19B is a dark field micrographs of clusters of
stem cells derived in the suspension culture system from the ICM of
a human in vitro fertilized blastocyst and expanded in the same
suspension culture conditions for 6 weeks with FIG. 19B being a
magnification of the marked section in FIG. 19A demonstrating the
typical morphological characteristics of undifferentiated hESC
clusters when cultured in suspension being compacted clusters of
uniformly small packed relatively transparent cells which do not
form cystic structures.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0090] The present disclosure provides a detailed description of
culture systems for handling suspensions of stem cells, preferably
human embryonic stem cells in an undifferentiated state. It should
be noted that in addition to the culture systems discussed in
detailed hereinbelow, also encompassed herein are uses of specific
components described with reference to the culture system in the
preparation of such stem cell cultures, as well as to methods of
use of the culture system in handling stem cell cultures and
methods of preparing cultured cells.
[0091] As used in the specification and claims, the forms "a", "an"
and "the" include singular as well as plural references unless the
context clearly dictates otherwise. For example, the term "a stem
cell" includes one or more stem cells, and the term "stem cells"
includes one stem cell as well as more than one stem cell.
[0092] As used herein, the term "or" means one or a combination of
two or more of the listed choices.
[0093] Further, as used herein, the term "comprising" is intended
to mean that the methods and culture systems includes the recited
elements, but does not exclude others. Similarly, "consisting
essentially of" is used to define methods and systems that include
the recited elements but exclude other elements that may have an
essential significance on the functionality of the culture systems
of the inventions. For example, a culture system consisting
essentially of a basic medium and medium supplements will not
include or will include only insignificant amounts (amounts that
will have an insignificant effect on the propagation of cells in
the culture system) of other substances that have an effect on
cells in a culture. Also, a system consisting essentially of the
elements as defined herein would not exclude trace contaminants.
"Consisting of" shall mean excluding more than trace amounts of
other elements. Embodiments defined by each of these transition
terms are within the scope of this invention.
[0094] Further, all numerical values, e.g., concentration or dose
or ranges thereof, are approximations which are varied (+) or (-)
by up to 20%, at times by up to 10%, from the stated values. It is
to be understood, even if not always explicitly stated that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
GLOSSARY
[0095] In the following description and claims use will be made, at
times, with a variety of terms, and the meaning of such terms as
they should be construed in accordance with the invention is as
follows:
[0096] "Stem cells", as used herein, refers to cells which under
suitable conditions are capable of differentiating into other cell
types having a particular, specialized function (i.e., "fully
differentiated" cells) while under other suitable conditions are
capable of self renewing and remaining in an undifferentiated
pluripotential state as detailed below. A "cell" as used herein
refers to a single cell as well as to a population of (i.e. more
than one) cells. The population may be a pure population comprising
one cell type. Alternatively, the population may comprise more than
one cell type. The stem cells are preferably, embryonic stem (ES)
cells obtained from the embryonic tissue formed after
fertilization, parthenogenetic activation or somatic cell nuclear
transfer (e.g., blastomer/s from cleavage stage embryo or morula,
blastocyst), or embryonic germ (EG) cells obtained from the genital
tissue of a fetus any time during gestation, preferably before 10
weeks of gestation, or induced pluripotent stem cells.
[0097] "Embryonic stem cell" and "Pluripotent embryonic stem cell",
as used herein, refer to a cell which can give rise to many
differentiated cell types in an embryo or an adult, including the
germ cells (sperm and eggs). This cell type is also referred to as
an "ES" cell.
[0098] "Cell culture" or "Cultured cell", as used herein, refer to
cells or tissues that are maintained, cultured, cultivated or grown
in an artificial, in vitro environment. Included within this term
are continuous cell lines (e.g. with an immortal phenotype),
primary cell cultures, finite cell lines (e.g., non-transformed
cells), and any other cell population maintained in vitro. In this
connection, a primary cell is a cell which is directly obtained
from a tissue or organ of an animal, including a human, in the
absence of culture. Typically, though not necessarily, a primary
cell is capable of undergoing ten or fewer passages in vitro before
senescence and/or cessation of proliferation.
[0099] "Undifferentiated pluripotential ES cells", "Pluripotent
SC", and "ESC", as used herein, refer to precursor cells that have
the ability to form any adult cell. Such cells are true cell lines
in that they: (i) are capable of indefinite proliferation in vitro
in an undifferentiated state; and (ii) are capable of
differentiation to derivatives of all three embryonic germ layers
(endoderm, mesoderm, and ectoderm) even after prolonged culture.
Human ES cells (hES cells) are derived from fertilized embryos that
are less than one week old (in the cleavage or blastocyte stage) or
produced by artificial means (such as by nuclear transfer) that
have equivalent characteristics.
[0100] "Undifferentiated", as used herein, refers to cultured cells
when a substantial proportion (at least 20%, and possibly over 50%
or 80%) of the cells and their derivatives in the population
display morphological characteristics of undifferentiated cells,
distinguishing them from differentiated cells of embryo or adult
origin. Cells are recognized as proliferating in an
undifferentiated state when they go through at least 1 population
doubling during a cultivation period of at least 3 weeks, while
retaining at least about 50%, or the same proportion of cells
bearing characteristic markers or morphological characteristics of
undifferentiated cells after said cultivation period.
[0101] "Maintenance" means continued survival of a cell or
population of cells, at times, with an increase in numbers of
cells. "Proliferation", "propagation", "expansion" and "growth",
which may be used interchangeably with each other, refer to such an
increase in cell number. According to one embodiment, this term
refers to a continuous survival of the cells for at least 6 weeks,
preferably for at least 10 weeks.
[0102] "Cell suspension" as used herein, refers to a culture of
cells in which the majority of the cells freely float in the
medium, typically a culture medium (system), and the cells floating
as single cells, as cell clusters and/or as cell aggregates. In
other words, the cells survive and propagate in the medium without
being attached to a solid or semi solid substrate.
[0103] "Culture system", as used herein, refers to culture
conditions for supporting the maintenance and propagation of SCs or
somatic cells derived therefrom, as well as, under selected
conditions, for supporting derivation and propagation of
embryos-derived clusters into hESC. The term denotes a combination
of elements, at minimum including a basic medium (a cell culture
medium usually comprising a defined base solution, which includes
salts, sugars and amino acids) and a serum replacement supplement.
The culture system may further comprise other elements such as,
without being limited thereto, an extracellular matrix (ECM)
component, additional serum or serum replacements, a culture
(nutrient) medium and other exogenously added factors, which
together provide suitable conditions that support SC growth. In the
relevant context, the term "culture system" also encompasses the
cells cultured therein.
[0104] The term "suspension supporting culture system" denotes
culture conditions, as explained above, which supports expansion of
SC in an undifferentiated pluripotent state, while the SC are in
suspension as well as which support derivation of embryos derived
clusters suspension into hESC and expansion of the latter. The
suspension supporting culture system comprises at minimum a basic
medium (a cell culture medium usually comprising a defined base
solution, which includes salts, sugars and amino acids) and a serum
replacement supplement (with respect to maintenance and expansion
in suspension of the SC, the most preferable SR is knock out serum
replacement (KOSR)). The culture system may further comprise other
elements such as, without being limited thereto, one or more of an
extracellular matrix (ECM) component, additional serum or serum
replacements, a culture (nutrient) medium and other exogenously
added factors, which together provide suitable conditions that
support SC growth. The conditions provided by the suspension
supporting culture system are such that SC can proceed through the
cell cycle, grow and divide while maintaining the morphology of
undifferentiated SC. Preferably, the conditions are such which
enable growth of human stem cells, preferably, human embryonic stem
cells (hESC). Further, the suspension supporting culture system
provides conditions that permit the SC to stably proliferate in the
culture system for at least 6 weeks, 10 weeks and even 20 weeks. It
is intended that the definition encompass outgrowth as well as
maintenance media.
[0105] The term "differentiation inducing culture system" denotes a
culture system, as explained above, which directs differentiation
of suspension derived SC into a somatic cells. The meaning of
"differentiation inducing culture system" should be read in
conjugation with the meaning of the term "culture systems that
supports differentiation into selected population of somatic
cells".
[0106] "Large scale", as used herein with regard to cell
cultivation and expansion, refers to the cultivation of SC under
conditions which permit at least the doubling of cells after 4
weeks. The term may be used to denote cultures of both
undifferentiated pluripotent stem cells and cultures of
differentiated cells derived from stem cells (either by directed
differentiation or by spontaneous differentiation).
[0107] "Long term" as used herein with regard to cell cultivation
and expansion, refers to the cultivation of SC for at least 6
weeks, preferably, for at least 10 weeks and more preferably, for
at least 20 weeks.
[0108] "Laminin-free culture system" refers to any culture system
which has not been supplemented with laminin or laminin equivalent
which being capable of providing culture conditions for the
maintenance and/or expansion of stem cells. A minimal amount of
laminin denotes not more than 10 ng/ml of laminin or laminin
equivalent.
[0109] "Cell marker", as used herein, refers to is any phenotypic
feature of a cell that can be used to characterize it or
discriminate it from other cell types. A marker may be a protein
(including secreted, cell surface, or internal proteins; either
synthesized or taken up by the cell); a nucleic acid (such as an
mRNA, or enzymatically active nucleic acid molecule) or a
polysaccharide. Included are determinants of any such cell
components that are detectable by antibody, lectin, probe or
nucleic acid amplification reaction that are specific for the cell
type of interest. The markers can also be identified by a
biochemical or enzyme assay that depends on the function of the
gene product. Associated with each marker is the gene that encodes
the transcript, and the events that lead to marker expression. A
marker is said to be preferentially expressed in an
undifferentiated or differentiated cell population, if it is
expressed at a level that is at least 5 times higher (in terms of
total gene product measured in an antibody or PCR assay) or 5 times
more frequently (in terms of positive cells in the population).
Markers that are expressed 10, 100, or 10,000 times higher or more
frequently are increasingly more preferred.
[0110] "Culture systems that supports differentiation into selected
population of somatic cells", as used herein, refers to a variety
of culture systems known in the art to promote specific
differentiation to a specifically desired population of somatic
cells. For example, a culture system that supports the directed
differentiation of SCs into neural precursor cells may comprise a
basic medium supplemented by FGF2 and/or noggin, as described, for
example by Itsykson, P., et al.sup.14. Further, for example, a
culture system that supports the directed differentiation into
dopaminergic neuronal cells will initially comprise the same
conditions supporting differentiation into neural precursor cells,
the latter directed into dopaminergic neuronal cells by the
supplementation of the medium with at least one of sonic hedgehog
(SHH), fibroblast growth factor (FGF), or a member of the Wnt
family.sup.15.
[0111] "Culture conditions/systems that support spontaneous
differentiation into somatic cells", as used herein, refers to any
culture conditions that promoted spontaneous nonspecific
differentiation of stem cells to a mixture of somatic cells from
any of the three embryonic germ layers: ectoderm; mesoderm; and
endoderm. The medium in such culture conditions will typically be
without components known to be required for the maintenance of SCs
in an undifferentiated (pluripotent) state. Such components
typically include soluble factors typically added to media for
maintenance of undifferentiated SCs. An example of a medium that
supports spontaneous differentiation of SCs into somatic cells
comprises a basic medium of DMEM supplemented by FCS 20%, as
described by Reubinoff et al..sup.1.
[0112] In its broadest sense, the present disclosure concerns
culture systems and methods for the maintenance and preferably
propagation of undifferentiated, pluripotent stem cells (SCs)
suspended in a culture system comprising basic medium and a serum
replacement that supports expansion of the suspended SC in an
undifferentiated pluripotent state. The culture system provided
herein has been found to be especially suitable for large scale and
long term maintenance of undifferentiated stem cells.
[0113] This culture system is referred to herein by the term
"suspension-supporting culture system". A preferred embodiment
encompasses a suspension-supporting culture system comprising a
basic medium and knockout serum replacement (KOSR). As indicated
above, it was surprisingly found that the combination of KOSR with
a basic medium provided conditions suitable for the expansion of
pluripotent SCs. The SCs were maintained in an undifferentiated
state for a long period, i.e. of at least 6 weeks, preferably, for
at least 10 weeks, more preferably, for at least 20 weeks.
[0114] The suspension-supporting culture system disclosed herein
allows the SCs to expand in the form of free floating
undifferentiated pluripotent SCs, free floating to clusters of
pluripotent undifferentiated SCs or free floating aggregates of
undifferentiated pluripotent SCs.
[0115] SCs can be obtained using well-known cell-culture methods.
For example, pluripotent stem cells may be obtained by inducing
reprogramming of somatic cells (induced pluripotent stem cells;
iPC) as described herein. Further, hESC can be isolated from human
blastocysts, morulas, cleavage stage embryos or blastomeres. Human
blastocysts are typically obtained from human preimplantation
embryos, from in vitro fertilized (IVF) oocytes,
parthenogenetically activated oocytes or following somatic cell
nuclear transfer. Alternatively, a single cell human embryo can be
expanded to the cleavage stage, morula or blastocyst stage. For the
isolation of human ES cells from blastocysts, most commonly the
zona pellucida is removed from the blastocyst. The whole blastocyts
may be used to derive stem cells or alternatively, the inner cell
mass (ICM) is isolated by immunosurgery, in which the trophectoderm
cells are lysed and removed from the intact ICM by gentle
pipetting. The ICM may be isolated by any alternative method
including mechanical methods or with the assistance of laser
[Turetsky T, Aizenman E, Gil Y, Weinberg N, Shufaro Y, Revel A,
Laufer N, Simon A, Abeliovich D, Reubinoff B E. Laser-assisted
derivation of human embryonic stem cell lines from IVF embryos
after preimplantation genetic diagnosis. Hum Reprod. 2008
January;23(1):46-53.]. The ICM, blastomeres or whole intact
blastocyte is then plated in a tissue culture flask containing the
appropriate medium which enables its outgrowth. Following 9 to 15
days, the outgrowth is dissociated into clumps either by a
mechanical dissociation or by an enzymatic degradation and the
cells are then re-plated on a fresh tissue culture medium. Colonies
demonstrating undifferentiated morphology are individually selected
by micropipette, mechanically dissociated into clumps, and
re-plated. Resulting ES cells are then routinely split every 1-2
weeks. For further details on methods of preparation human ES cells
see Thomson et al. [U.S. Pat. No. 5,843,780; Science 282:1145,
1998; Curr. Top. Dev. Biol. 38:133, 1998; Proc. Natl. Acad. Sci.
USA 92: 7844, 1995]; as well as Bongso et al. [Hum Reprod 4: 706,
1989]; Gardner et al. [Feral. Steril. 69:84, 1998]; and Klimanskaya
et al. [Nature. 446: 342, 2007].
[0116] Commercially available SCs can also be used in accordance
with the invention. hESCs can be purchased from the NIH human
embryonic stem cells registry. Non-limiting examples of
commercially available embryonic stem cell lines are BG01, BG02,
BG03, BG04, CY12, CY30, CY92, CY10, TE03 and TE32.
[0117] Pluripotent SCs present at their surface, or express,
biological markers which are used to identify pluripotent SCs as
well as to verify that the cells in the culture are maintained in
an undifferentiated state [Thomson J A et al. Embryonic Stem Cell
Lines Derived from Human Blastocysts Science 282(5391):1145-1147
(1998)]. A non-limiting list of such cell markers comprise
stage-specific embryonic antigens such as SSEA-3 and SSEA-4;
antibodies to specific extracellular matrix molecule which are
synthesized by undifferentiated pluripotent SC, such as TRA-1-60,
TRA-1-81, and GCTM-2; elevated expression of alkaline phosphatase,
which is associated with undifferentiated pluripotent SCs; and
transcription factors unique to pluripotent SCs and which are
essential for establishment and maintenance of undifferentiated
SCs, such as OCT-4, Nanog and Genesis [Carpenter, M. K., Rosier,
E., Rao, M. S., Characterization and Differentiation of Human
Embryonic Stem Cells. Cloning and Stem Cells 5, 79-88, 2003].
[0118] Generally, the basic medium may be any basic medium known in
the art for culturing cells, in particular, stem cells. In a
preferred embodiment the basic medium is selected from
Neurobasal.TM. [Cat. No. 21103-049 Gibco 1998/1999], Cellgro Stem
Cell Growth Medium [Cat No. 2001 CellGenix Germany 2005], KO-DMEM
[Cat. No, 10829-018 Gibco 1998/1999] and X-Vivo 10 [Cat. No.
04-380Q Lonza Switzerland 2007]. Most preferably the present
invention makes use of Neurobasal.TM. as the basic medium (i.e. the
basic media consists essentially of Neurobasal.TM.). Neurobasal.TM.
is known in the art of cell cultures [Brewer G J. Serum-free
B27/Neurobasal medium supports differential growth of neurons from
the striatum, substantia nigra, septum, cerebral cortex, J Neurosci
Res. 42(5):674-83, (1995)] and is commercially available [Gibco,
Invitrogen cell culture, USA].
[0119] The suspension-supporting culture system, e.g.
Neurobasal.TM. supplemented with KOSR, may be further supplemented
by other components known to be used in culture systems,
comprising, without being limited thereto, a member of FGF family.
In accordance with one embodiment, the FGF member is, without being
limited thereto, FGF2.
[0120] The suspension-supporting culture system may be further
supplemented by an extracellular matrix (ECM) component. In
accordance with one embodiment, the ECM is selected from, without
being limited thereto, fibronectin, laminin and gelatin.
[0121] The suspension-supporting culture system may be further
supplemented by an antibacterial agent. The antibacterial agent may
be selected from, without being limited thereto, penicillin and
streptomycin.
[0122] The suspension-supporting culture system may be further
supplemented by non-essential amino acids (NEAA).
[0123] In addition, the suspension-supporting culture system may be
further supplemented by a TGF.beta. superfamily factor. The
TGF.beta. superfamily factor may be, without being limited thereto,
activin A.
[0124] The suspension-supporting culture system may be further
supplemented by a neurotrophin. Neutrophins are known to play a
role in assisting to promote the survival of SCs in culture. In
accordance with an embodiment of the invention, the neurotrophin is
selected from, without being limited thereto, BDNF, NT3, NT4.
[0125] The suspension-supporting culture system may be further
supplemented by nicotinamide (NA). It is noted that NA may assist
in preventing the differentiation of cells into extraembryonic
lineages, maintaining them as undifferentiated cells, assist in
promoting the cells' survival and proliferation (WO 03/104444).
[0126] The suspension-supporting culture system may be further
supplemented by a bone morphogenic protein (BMP) antagonist. It is
noted that under culture conditions that support undifferentiated
proliferation of hESCs noggin (a BMP antagonist) prevents
extraembryonic background differentiation of hESCs. While under
conditions that promote differentiation, noggin is known to prevent
the differentiation to non-neural lineages, favoring the
differentiation to a neural fate. The BMP antagonist may be
selected from, without being limited thereto, noggin, chordin, or
gremlin.
[0127] Further, the suspension-supporting culture system may be
supplemented by a serum free medium supplement. The serum free
medium supplement may be selected from, without being limited
thereto, Nutridoma-CS or TCH.TM. [Catalog No. 3001, Protide
Pharmaceuticals]. In accordance with a preferred embodiment, the
suspension-supporting culture system is supplemented by
Nutridoma-CS.
[0128] In accordance with one preferred embodiment, the method
disclosed herein makes use of the suspension-supporting culture
system for the expansion, in a suspension, of embryonic SCs. A
further preferred embodiment disclosed herein encompasses the
expansion in a suspension of human SCs (hESC).
[0129] A further preferred embodiment concerns the method disclosed
herein making use of suspension-supporting culture system for the
expansion, in an undifferentiated state, of pluripontent hESCs.
Most preferably, the suspension-supporting culture system is used
for the expansion of hESCs. To this end, the suspension-supporting
culture system comprises Neurobasal.TM. and KOSR, the
suspension-supporting culture system permits expansion of the cells
in an undifferentiated pluripotent state. Most preferably, the
suspension-supporting culture system comprises a basic medium
consisting of Neurobasal.TM. and further comprising KOSR.
[0130] The results presented hereinbelow also show that the
suspension-supporting culture system may be free of laminin or
include minimal amount of laminin (not more than 10 ng/ml). Laminin
has been shown in culture to stimulate neurite outgrowth, promote
cell attachment, chemotaxis, and cell differentiation. It has now
been found that when supplementing the basic media with a serum
free medium supplement, e.g. Neutridoma CS, not only there is no
need to add laminin but also, an increase number of cells was
observed after a long term (six weeks) of cultivation in
suspension, as compared to the number of cells outgrown in the
presence of laminin. For longer culture periods, a minimal
concentration (reduced by 1000 times; namely, not more than 10
ng/ml was sufficient for propagation and expansion of the cells for
at least 20 weeks.
[0131] The suspension-supporting culture system is preferably
suitable for long term expansion of the SCs suspended therein. In
accordance with one embodiment, long term refers to at least six
weeks of cultivation and cell expansion. In accordance with another
embodiment, long term refers to a period of at least 10 weeks. In
accordance with yet another embodiment, long term refers to a
period of at least 20 weeks. During this term, the SCs exhibit cell
markers which confirm that the SCs are essentially maintained in an
undifferentiated pluripotent state.
[0132] It should be well appreciated by those versed in the art
that the suspension-supporting culture system with which the SCs
are maintained for a long period of time in an undifferentiated,
pluripotent state, may be of significant benefit for large scale
propagation of SCs in bioreactors. As well appreciated by those
versed in the art, it is advantageous to provide conditions that
allow for large scale production of standardized homogeneous
undifferentiated hESCs. The advantages of suspension culture
bioreactors were also described above, in particular for large
scale expansion.
[0133] A preferred embodiment disclosed herein concerns a method of
expanding SCs in an undifferentiated pluripotent state, the method
comprising incubating undifferentiated pluripotent SCs in
suspension within a culture system comprising basic medium and
knockout serum replacement (KOSR). Preferably, the method comprises
incubating undifferentiated SCs in suspension within a culture
system comprising basic medium consisting of Neurobasal.TM. and
further comprising knockout serum replacement (KOSR).
[0134] The methods disclosed herein also comprise the step of
obtaining SCs from SC colonies cultivated on a feeder layer or in a
feeder free adherent culture system. The SCs may be obtained by
dissociation of the cells from the culture system, e.g. by the aid
of suitable agents (e.g. collagenase IV), trituration and
transferring the dissociated SCs into the culture system of the
invention to form a suspension of SCs, the latter being in the form
of free floating cells, free floating clusters of cells or free
floating aggregates of cells.
[0135] The methods may also comprise one or more steps of media
refreshment (i.e. the replacement of at least 50% of the culture
system). It is appreciated that by conducting said media
refreshment, dead cells and their fragments are gradually removed.
Culture media may be refreshed at least every 2-3 days, and most
preferably at least every 2 days. The media refreshment may include
the replacement of a portion of the basic media only, as well as
the replacement of a portion of the basic media including one or
more of its components as described above. Further, it is
appreciated that the methods may comprise different media
replacements, e.g. at times only the replacement of the basic
medium, and at other time points, the replacement of the basic
medium comprising one or more of the supplements.
[0136] It is also appreciated that as a result of cells expansion,
the SCs may proliferate into large clusters. Thus, the methods
disclosed herein may also comprise one or more SCs manipulations so
as to disaggregate the big clusters of cells resulting from their
overgrowth. According to one embodiment, the overgrowth of the
cells in clusters is prevented by trituration. The essentially
disaggregated cells may then be transferred to suitable tissue
culture carriers (e.g. dishes, culture tubes, culture bioreactors,
etc.) for continued expansion. Overgrowth of clusters may be
prevented by other means such as chopping the clusters or the use
of increased shearing forces of bioreactor systems or any other
method known in the art.
[0137] It has been found that the undifferentiated and pluripotent
SCs obtained using the suspension-supporting culture system
disclosed herein may be, at any stage of expansion, induced to
differentiate into a variety of somatic cells from the three
embryonic major lineages; endoderm, ectoderm and mesoderm. For
differentiation induction, the undifferentiated and pluripotent SCs
are transferred to a culture system supporting differentiation.
Such culture system is referred to herein by the term
"differentiation inducing culture system".
[0138] Thus, in accordance with another aspect, there are provided
the use of undifferentiated pluripotent SC in suspension in methods
for directing differentiation of SCs from suspension culture
systems into a selected population of somatic cells, the method
comprising:
[0139] (a) providing a suspension of undifferentiated SCs
comprising a basic medium and a serum replacement supporting SC in
suspension, preferably KOSR; and
[0140] (b) culturing the SC in a differentiation inducing culture
system that supports directed differentiation of SCs into the
selected population of somatic cells, namely, a dedicated
differentiation inducing culture system.
[0141] In other words, there is disclosed herein the use of the
undifferentiated and pluripotent SC expandable in suspension for
the preparation of a composition comprising a selected SC-derived
population of somatic cells.
[0142] Depending on the specific media composition of the
differentiation inducing culture system, the nature of the
SC-derived population may be a priori determined. In other words,
the undifferentiated and pluripotent SCs obtained from the
suspension-supporting culture system of the SC may be induced to
differentiate into a specific and pre-selected fate. To this end,
the undifferentiated and pluripotent SCs are cultivated in a
culture system (the cells with the differentiation inducing culture
media) that directs differentiation to a specifically desired
population of somatic cells, thereby providing a population of
cells highly enriched for a specific cell type or a pure population
of cells of a single type. A variety of single type somatic cell
populations may be derived from undifferentiated and pluripotent
SCs and those versed in the art will know how to select the medium
components and establish the desired differentiation inducing
culture system which directs the specific differentiation of the
latter to the desired population of somatic cells.
[0143] For example, directing differentiation of undifferentiated
and pluripotent SCs to neural precursor cells or neural stem cells
may be obtained by cultivating the SCs in a differentiation
inducing culture system comprising DMEM/F12 medium (Gibco)
supplemented with B27 (1%, Gibco) (DMEM/F12/1327 medium), FGF-2 (20
ng/ml) and noggin (750 ng/m, R&D Systems, Inc., Minneapolis,
Minn.) as exemplified hereinbelow and also by Itsykson, P., et
al..sup.(14) or by Reubinoff et al..sup.(24).
[0144] Further, for example, directing differentiation of SCs to
midbrain dopamineric neuronal cells may be obtained by first by
inducing differentiation into neural precursor cells, such as
described above, followed by cultivation of the neural precursor
cells in a basic medium, such as DMEM/F12/B27 medium supplemented
with at least one of fibroblast growth factor, preferably
fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), a
member of the Wnt family, preferably Wnt 1, as exemplified below
and also described by Yan, Y. et al..sup.15. Co-culture with cells
that promote midbrain differentiation such as the PA6 stromal
cells, or midbrain astrocytes may be also used. The resulting cells
may be further differentiated into midbrain dopaminergic neurons in
the presence of one or more of a member of the Wnt family, such as,
Wnt5a, at least one or more of an FGF, such as FGF20, and any one
of dibutyryl cyclic AMP (dbCAMP), glial cell derived neurotrophic
factor (GDNF), transforming growth factor .beta.3 (TGF.beta.3),
ascorbic acid, Neurotrophin 3 and 4 (NT3 and NT4).
[0145] to The undifferentiated and pluripotent SC may be directed
to differentiate into any other sub-type of peripheral or central
nervous system neurons or glia cells including, without being
limited thereto, gabaergic and glutamaergic nerons, first by
inducing differentiation into neural precursor cells, such as
described above, followed by cultivation of the neural precursor
cells in a basic medium, such as DMEM/F12/B27 medium supplemented
with differentiation inducing factors and survival promoting
factors such as ascorbic acid, NT3 and NT4
[0146] In accordance with another aspect disclosed herein, the
undifferentiated and pluripotent SCs obtained from the
suspension-supporting culture system may be induced for spontaneous
non-specific differentiation of somatic cells.
[0147] Thus, there is also provided herein the use of
undifferentiated and pluripotent SC in expandable in suspension in
a method for promoting spontaneous differentiation of SCs into
somatic cells, the method comprising:
[0148] (a) providing a suspension of undifferentiated SCs in a
culture system comprising a basic medium and a serum replacement
that supports expansion of said SC in a suspension, preferably
knockout serum replacement (KOSR);
[0149] (b) incubating said SCs in culture system that support
spontaneous differentiation of SCs into the somatic cells.
[0150] The spontaneous differentiation of SCs into the somatic
cells may be achieved, for example, by the use of a culture system
that is free of soluble factors and/or ECM components typically
used for maintaining SCs in undifferentiated state, the exclusion
of soluble factors and/or ECM components from the culture system is
known to promote spontaneous differentiation of SCs, as also
described by Itsykson, P., et al..sup.(14) Reubinoff et al [Nat
Biotechnol 18, 399-404 (2000)]. A culture system that is free of
soluble factors and/or ECM components is referred to herein by the
term "spontaneous differentiation inducing culture system".
[0151] In accordance with the above method for "spontaneous
differentiation" of suspension cultured SC, the undifferentiated
pluripotent SCs (in suspension) are firstly induced to form
embryoid bodies (EBs) by transferring and culturing the clusters in
KO DMEM, supplemented with fetal bovine serum in the absence of
soluble factors and extracellular matrices. The said EBs are then
disaggregated and further plated and cultured in the same medium of
the EBs. It is noted that the production of a mixture of
non-specific somatic cells was evident by expression of human
muscle actin, indicating the presence of mesodermal cells in the
cell culture; or by expression of Sox-17, indicating the presence
of endodermal cells in the cell culture.
[0152] Methods other than spontaneous differentiation within EBs
may be used to derive somatic cells, for example differentiation in
flat culture; and other culture systems may be used to promote
differentiation towards specific cell types such as those described
by D'Amour K A et al. [D'Amour K A Production of pancreatic
hormone-expressing endocrine cells from human embryonic stem cells.
Nat Biotechnol. 2006 November; 24(11):1392-401)] or by Yao S. et
al. [Yao S, Long-term self-renewal and directed differentiation of
human embryonic stem cells in chemically defined conditions. PNAS
2006; 103: 6907-6912].sup.(26,27).
[0153] It is noted that the undifferentiated pluripotent SCs
maintained in suspension, as disclosed herein, may also be used for
the production of Teratoma tumors. This may be achieved by
injection of the pluripotent SCs into an animal. As may be
appreciated, the formation of teratoma tumors evidences that the
formed cells are pluripotent cells.
[0154] As indicated above, the suspension-supporting culture system
is especially suitable for large scale production of SCs. Thus, it
should be appreciated that the methods disclosed herein for the
production of specific as well as non-specific somatic cells,
making use of the SCs produced using the culture system of the
invention, are also suitable for large scale production of such
somatic cells. In other words, the present disclosure provides
methods for the large scale production of SCs as well as for the
large scale production of somatic cells derived therefrom (either
by spontaneous or directed differentiation).
[0155] Finally, there is disclosed herein a method for deriving
stem cells in suspension from human embryos the method comprising:
[0156] (a) providing in vitro fertilized embryos; [0157] (b)
culturing said embryos to a blastocyte stage; [0158] (c) isolating
from said blastocyte inner cell mass (ICM); [0159] (d) culturing
said ICM in suspension within a feeder free culture system
comprising a non-adherent culture dish, and one or more of a basic
media, serum a replacement, an extra cellular matrix and a soluble
factor that promote the maintenance of pluripotent stem cells until
clusters are formed; [0160] (e) allowing said clusters to propagate
and expand;
[0161] said clusters and cells within them exhibiting morphological
characteristics of clusters of undifferentiated hluripotent hESC
when cultured in suspension.
[0162] A specific morphological characteristic comprises compacted
clusters of uniformly small packed, relatively transparent cells,
which do not form cystic structures.
[0163] Further, anmorphological characteristic of hESCs may
comprise, without being limited thereto, expression of genes SSEA4,
TRA1-60, TRA1-81, OCT-4, nanog, Rex-1, and TERT (markers of
pluripotency).
[0164] In addition, a morphological characteristic of hESCs may
comprise non-expression of the genes PSA-NCAM (marker for neural
cell); FGF-5 (related to early ectodermal differentiation neural
precursors); PAX-6 and nestin; 3CB2 (markers for radial glia/neural
stem) .beta.-III tubulin and thyrosine hydroxilase (TH), (markers
for dopaminergic), GABA (marker for gabaergic) and glutmate (marker
for glutamaergic); and enlagraid-1 (the co-expression of which with
TH being a marker for midbrain dopaminergic neurons).
[0165] It is appreciated that certain features disclosed herein,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the present disclosure,
which are, for brevity, described in the context of a single
embodiment, may also be provided separately or in any suitable
sub-combination.
[0166] Although the present disclosure has been described in
conjunction with specific embodiments thereof, it is evident that
many alternatives, modifications and variations will be apparent to
those skilled in the art.
[0167] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification.
[0168] The invention will now be described with reference to the
following non-limiting examples.
SOME EXEMPLARY EMBODIMENTS
Materials and Methods
Human Feeders and Preparation of Feeder Layers
[0169] Foreskin cells from an established line were used as feeders
as previously described (Banin E, Obolensky A, Idelson M, Hemo I,
Reinhardtz E, Pikarsky E, Ben-Hur T, Reubinoff B. Retinal
incorporation and differentiation of neural precursors derived from
human embryonic stem cells. Stem Cells 2006; 24(2): 246-57).
Briefly, the foreskin fibroblasts were cultured in DMEM (Gibco,
Gaithersburg, Md.) supplemented with 10% Fetal Calf Serum (FCS)
(Biological Industries, Beit Haemek, Israel). They were passaged by
trypsin (Gibco) digestion. For the preparation of feeder layers
3.times.10.sup.5 cells were plated per well of a six well plate
(Corning, N.Y., USA), precoated with 0.1% gelatin (Sigma, St.
Louis, Mo.). Mitotic inactivation of the feeders was carried out
prior to plating by incubating them 2.5 hours with Mitomycin-C 5
.mu.g/ml (Kyowa, Tokyo).
hESC Culture System
[0170] hESCs were cultured on the human feeder layers in KO medium
(KOM) consisting of 85% KO-DMEM, 15% KOSR, 1 mM glutamine, 0.1 mM
.beta.-mercaptoethanol, 1% nonessential amino acids, 50 units/ml
penicillin, 50 .mu.g/ml streptomycin, (Gibco, Gaithersburg, Md.)
and 4 ng/ml FGF-2 (R&D Systems, Inc., Minneapolis, Minn.).
hESCs were weekly passaged with Ca/Mg.sup.++-free PBS supplemented
with 0.05% EDTA (Biological Industries, Beit Haemek, Israel) or
type IV collagenase (1 mg/ml; Gibco) and plated onto fresh feeder
layer.
Suspension Culture of hESC hESC colonies that were cultivated on
human feeders in the KO culture system described above and in WO
2006/070370, the entire contents of which is specifically
incorporated herein by reference, were firstly dissociated with the
aid of collagenase IV (1 mg/ml, 2-3 hours at 37.degree.). Cell
dissociation was promoted by agitation of the culture plate. The
cell clusters obtained were resuspended in fresh NBSR medium
(Neurobasal.TM., 14% KOSR, glutamine 2 mM, 50 units/ml penicillin,
50 .mu.g/ml streptomycin, 1% nonessential amino acids; Gibco), or
where indicated in fresh NBN2 medium (Neurobasal.TM., N2 supplement
1:100, glutamine 2 mM, 50 units/ml penicillin, 50 .mu.g/ml
streptomycin) and transferred to non-adherent culture dishes (Cell
Seed HydroCell). Both media were supplemented with FGF-2 20 ng/ml,
activin 25 ng/ml, fibronectin and laminin 5 .mu.g/ml each, gelatin
0.001%, and BDNF, NT3 and NT4, 10 ng/ml each.
[0171] When indicated, cell clusters were resuspended in NBSR
medium supplemented with 1.times. Nutridoma-CS (Roche, Germany Cat.
No. 1363743; 2007), FGF-2 20 ng/ml, activin 25-50 ng/ml,
fibronectin 5 .mu.g/ml, and gelatin 0.001%, with and without
laminin 5 .mu.g/ml.
[0172] The suspension was either strained though 30-50 micron mesh
to remove big clumps, or triturated by pipetting to disaggregate
big clumps and transferred into tissue culture dishes (Costar.RTM.,
Corning Inc., Corning, N.Y.) at a density of
.about.0.7-1.2.times.10.sup.6 cells/ml. Dead cells and their
fragments were gradually removed during media refreshment every two
days. The cells proliferated as free-floating tiny transparent
clusters of 20-50 cells. Aggregation and overgrowth of clusters was
prevented by trituration with a 1000 .mu.l pipettor tip as
required.
Characterization of hESC Grown in Suspension by FACs Analysis
[0173] For characterization of the cells within the small
free-floating aggregates, hESCs were dissociated with a solution of
2.25 mM EDTA with 0.06% trypsin, for 7-10 min at 37.degree.,
followed by gentle trituration, to obtain a single-cell suspension
solution.
[0174] The hESC were then washed with FACS media consisting of PBS
supplemented with 1% BSA and 0.05% sodium azide. The single-cell
suspension was stained with anti-SSEA4 (1:100, mouse monoclonal
IgG3, Developmental Studies Hybridoma Bank (DHSB), Iowa City,
Iowa), anti-Tra-1-60 (1:100, monoclonal mouse IgM, Chemicon
International), anti-Tra-1-81 (1:100, monoclonal mouse IgM,
Chemicon International), to anti-SSEA3 (1:100, monoclonal rat IgM,
Chemicon International) and anti-PSA-NCAM (1:100, monoclonal mouse
IgM, Chemicon International). Control hESCs were stained with their
respective isotype control antibodies. Primary antibodies were
detected using fluorescein isothiocyanate (FITC)-labeled goat
anti-mouse Ig (1:100, Dako) or Alexa Fluor-labeled goat anti-rat
IgM (1:100, Invitrogen). Propidium iodide (PI) was added (final
concentration of 4 .mu.g/ml) for better gating of viable cells.
FACS analysis was performed using the FACSCalibur system
(Becton-Dickinson, San Jose, Calif.).
Replacing and Monolayer Culture of hESCs Cultivated in
Suspension
[0175] Floating aggregates of hESCs were triturated with a 1000
.mu.l pipettor tip. Tiny clusters that were obtained were plated on
fresh feeders and cultured in KO DMEM medium supplemented with, 14%
KOSR, glutamine 2 mM, 50 units/ml penicillin, 50 .mu.g/ml
streptomycin, 1% nonessential amino acids; Gibco) supplemented with
4 ng/ml FGF2. After 1 week, colonies with typical morphological
characteristics of the colonies of undifferentiated hESCs,
developed on the feeders. These colonies could be passaged
routinely as described before. Alkaline phosphatase activity of the
cells within the plated colonies was demonstrated using the
Alkaline Phosphatase Substrate kit I by Vector Laboratories
(Burlingame, Calif.) according to the manufacturer's
instructions.
EB Formation
[0176] The clusters of undifferentiated hESCs were transferred and
cultured for 3-4 weeks in DMEM (Gibco), supplemented with 20% FBS
(Biological Industries, Beit Haemek), 1 mM L-glutamine, 0.1 mM
.beta.-mercaptoethanol, 1% non-essential amino acid stock, 50
units/ml penicillin, 50 .mu.g/ml streptomycin (all from Gibco
Invitrogen Corporation products, USA).
Induction of Somatic Differentiation and Immunohistochemistry
[0177] Clusters of undifferentiated hESCs were transferred and
cultured for 2-3 weeks in suspension within bacteriological dishes
precoated with 0.1% low melting temperature agarose in DMEM/F12
medium (Gibco) supplemented with B27 (1%, Gibco) (DMEM/F12/B27
medium), FGF-2 (20 ng/ml) and noggin (750 ng/m, R&D Systems,
Inc., Minneapolis, Minn.).
[0178] Clumps of neural precursors were triturated to small
clusters and plated on poly-D-lysine (30-70 kDa, 10 .mu.g/ml;
Sigma, St. Louis, Mo.) and laminin-coated (4 .mu.g/ml; Sigma) glass
coverslips and cultured for an additional week with DMEM/F12/B27
medium in the absence of growth factors.
[0179] In addition, to promote differentiation to midbrain
dopaminergic neurons, clumps of neural precursors were triturated
to small clusters and plated on poly-D-lysine and laminin-coated
glass coverslips (as above) and cultured for 2 weeks with
DMEM/F12/B27 medium supplemented with FGF8 and SHH.
[0180] EBs were dissociated with the aid of trypsin (0.025%, 3 mM
EDTA in PBS) digestion, and plated on poly-D-lysine and laminin
pre-coated glass coverslips (as above) and cultured for additional
1-2 weeks in the culture medium used for induction of
differentiation of EBs.
[0181] Differentiated cells within the outgrowth were fixed with 4%
paraformaldehyde for 20 minutes at room temperature. Cell membranes
were permeabilized with 0.2% Triton X100 (Sigma) in PBS for 5
minutes for immunostaining with anti-intracellular marker
antibodies. Neural precursors were incubated with the following
primary antibodies: anti-.beta.-III-tubulin (mouse monoclonal
IgG2b, 1:2000, Sigma), anti-rabbit TH (1:200, Pel Freeze), and
anti-mouse EN-1 (1:100, Developmental Studies Hybridoma Bank
(DHSB), Iowa City, Iowa). Mesodermal differentiation within EBs'
outgrowth was detected with the antibody against human muscle actin
(1:50, DAKO). Endodermal differentiation within EBs' outgrowth was
detected with the antibody against Sox-17 (1:50, R&D Systems
Inc.). Primary antibody localization was performed by using
fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit
immunoglobulins (Dako, 1:20-50), or goat anti-mouse immunoglobulin
conjugated with Cy3 (1:500 Jackson ImmunoResearch
Laboratories).
Teratoma Formation in NOD/SCID Mice
[0182] Clumps of hESCs that were propagated for 7 weeks in
suspension were injected into the testis of six weeks old NOD/SCID
mice (Harlan, Jerusalem, Ill.) (30-40 clumps per testis). Eight to
twelve weeks later, the resulting tumors were removed, embedded in
paraffin and sections were stained with H&E.
Stem Cells Derivation
[0183] The inner cell mass (ICM) of preimplantation genetic
diagnosed in vitro fertilized 5 blastocysts was isolated by a
laser-assisted system [Turetsky T, Aizenman E, Gil Y, Weinberg N,
Shufaro Y, Revel A, Laufer N, Simon A, Abeliovich D, Reubinoff B E.
Laser-assisted derivation of human embryonic stem cell lines from
IVF embryos after preimplantation genetic diagnosis. Hum Reprod.
2008 January; 23(1):46-53]. ]) and transferred to the
suspension-supporting culture conditions as disclosed above
(Neurobasal.TM., 14% KOSR, 1.times. Nutridoma-CS, glutamine 2 mM,
50 units/ml penicillin, 50 .mu.g/ml streptomycin, 1% nonessential
amino acids; FGF-2 20 ng/ml, activin 25 ng/ml, laminin 10 ng/ml,
gelatin 0.001%, and BDNF, NT3 and NT4, 10 ng/ml each. The ICM was
cultured within this medium in nonadherent culture dishes. Rock
inhibitor (Sigma) was added to the culture medium during the first
two days. The cells from one of the ICMs proliferated leading to an
increase in the size of the cluster and generating new clusters.
The clusters of cells were passaged by mechanical dissection.
Results
[0184] NBSR Suspension Culture System for the Propagation of hESC
in Bulk
[0185] To develop suspension cultures, hESC colonies that were
cultivated on human feeders in the KO culture system were
dissociated with the aid of type N collagenase. The cells/cell
clusters that were obtained were re-suspended within fresh NBSR
medium, supplemented with FGFs (FGF-2 20 ng/ml).
[0186] Further supplementation of the medium with one or more of
the following components increased the survival/proliferation and
prevented differentiation of the cells: [0187] TGF.beta.
superfamily factors (e.g., activin 25-50 ng/ml) [0188] ECM
components (e.g., laminin 5 .mu.g/ml, fibronectin 5 .mu.g/ml,
gelatin 0.001%) [0189] Neurotrophins (e.g., NT4, NT4, BDNF 10 ng/ml
each)
[0190] The cells were transferred to suspension at a density of
-0.7-1.2.times.10.sup.6 cells/ml. Dead/fragmented cells were
gradually removed during medium refreshment. The cells proliferate
as free-floating tiny transparent clusters of 20-50 cells without
any morphological signs of differentiation (FIG. 2). Aggregation
and overgrowth of the transparent clusters were prevented by
trituration through a 1000 .mu.l pipette tip as required. The hESCs
grown in a suspension comprising Neurobasal.TM. medium supplemented
with KOSR (NBSR) were sub-cultured by mechanical
disaggregation.
[0191] The cells expressed SSEA4, TRA1-60, and TRA1-81 (markers of
pluripotency), and did not express markers of somatic
differentiation such as the neural marker PSA-NCAM. Specifically,
after 3 weeks of cultivation in suspension under these culture
conditions, >90% of the cells expressed SSEA4, TRA1-60, and
TRA1-81 but not PSA-NCAM (FIGS. 3A-3E).
[0192] When re-plated on human feeders, after 3 and 7 weeks of
suspension culture, the cells gave rise to monolayer colonies with
the morphology of undifferentiated hESCs (FIG. 4A). The stem cells
within these colonies expressed alkaline phosphatase (a marker used
to identify stem cells; FIG. 4B).
[0193] It was concluded that efficient propagation of
undifferentiated hESC in suspension is achievable using the unique
combination of Neurobasal.TM. medium as the basic medium of the
culture system, supplemented with KOSR (NBSR).
[0194] Suspension in NBSR was shown to be more effective than
suspension in NBN2 (Neurobasal.TM. supplemented with N2) for the
expansion of hESCs without differentiation. Specifically, after 3
weeks, the total number of cells in each of the cell cultures (NBSR
vs. NBN2) was determined using trypan blue to include only live
cells and hESCs propagated in NBSR reached a population of
approximately double that of NBN2-cultured hESCs (FIG. 1).
[0195] NBSR medium that was supplemented with Nutridoma-CS, FGF-2,
activin, ECM components (fibronectin, gelatin and laminin), and
neutrophins could effectively support long-term cultivation of
undifferentiated hESCs. The percentage of hESCs expressing markers
of pluripotent stem cells (SSEA4, TRA1-60, and TRA1-81) was high
(>95%) and stable after 3.5 weeks and 6.5 weeks of suspension
culture (FIG. 5) as well as after 7 and 20 weeks (FIGS. 15 and 18).
Moreover, the cells expressed genes of pluripotency such as OCT-4,
nanog, Rex-1, and TERT and did not express the gene FGF-5, related
to early ectodermal differentiation (FIG. 16).
[0196] Furthermore, NBSR medium supplemented with Nutridoma-CS did
not require the addition of laminin. Using the same supplementation
as described above, however, with or without laminin showed that
the percentage of undifferentiated cells cultured in
NBSR/Nutridoma-CS was not significantly different in
laminin-positive cultures (+LAM) versus cultures in which laminin
was not added (-LAM) (FIG. 6). All the more and quite surprisingly,
in the absence of laminin, an increased number of cells was
observed after long-term cultivation (6.5 weeks) in the above
described NBSR/Nutridoma-CS culture suspension (FIG. 7). For longer
culture periods, a minimal concentration (reduced by 1000 times; 10
ng/ml or less) was sufficient for propagation and expansion for at
least 20 weeks.
Induced Differentiation of hESCs into Somatic Cells In Vitro
[0197] Undifferentiated hESCs that were cultivated in suspension
within NBSR gave rise upon differentiation to somatic progeny.
[0198] To induce neural differentiation the clusters were
transferred and cultured for 2 weeks in DMEM/F12 supplemented with
B27 (1%), FGF-2 (20 ng/ml) and noggin (750 ng/ml). These culture
conditions had been previously demonstrated to be highly efficient
for induction of neural differentiation of hESCs.sup.(14). The
clusters were then plated on laminin-coated slides and cultured for
a week in the same medium in the absence of growth factors. Cells
with morphological characteristics of neurons (FIG. 8A) gradually
migrated from the clusters. Immunostaining demonstrated that these
cells expressed markers of neural precursors PAX-6 and nestin (FIG.
17A), neural stem/radial glia cells (FIG. 17B, 3CB2), and neurons
(FIGS. 8B and 8C), including subtypes of neurons such as
dopaminergic (FIG. 17C, .beta.-III tubulin and thyrosine
hydroxilase (TH)), gabaergic (FIG. 17D, GABA) and glutamaergic
(FIG. 17E, glutmate). When the clusters were plated on laminin
coated slides for 2 weeks of differentiation in the presence of
FGF8 and SHH (which promote differentiation toward a midbrain
dopaminergic neuronal fate), multiple neurons co-expressing
enlgraid-1 (EN-1) and tyrosine hydroxylase (TH) were observed (FIG.
9). The co-expression of these markers is characteristic of
midbrain dopaminergic neurons.sup.15. The arrow shows a cell which
co-expresses EN-1 and TH. The arrow is hard to see on the B/W
figure. On the B/W scale, TH is indicated by light gray, and EN-1
is indicated by darker gray.
[0199] To induce mesodermal and endodermal differentiation the
clusters were transferred to DMEM supplemented with 20% FBS where
they formed embryoid bodies (EBs). After 3-4 weeks of
differentiation the EBs were dissociated, plated and cultured for
1-2 weeks on laminin coated coverslips. Immunostaining showed
differentiated cells expressing muscle actin (mesoderm; FIG. 10),
and Sox 17 (endoderm; FIG. 11).
[0200] To further demonstrate that the hESCs retained their
pluripotent potential after cultivation in suspension, clusters of
hESCs that were cultured in suspension for 7 weeks were injected
into the testis of NOD/SCID mice. Teratoma tumors were removed
after 8 weeks and histological analysis of the tumors demonstrated
differentiated progeny of the three embryonic germ layers (FIGS.
13A-13C). Formation of teratoma is a key feature of hESCs and is an
important evidence that the cells that grow in suspension are
indeed pluripotent cells.
The Suspension Culture System Support Derivation of New Stem Cell
Lines from Human Blastocysts
[0201] To derive new stem cell lines from human blastocysts, in
vitro fertilized embryos that were found in preimplantation genetic
diagnosis (PGD) to carry a mutation in BRCA1 and to be affected by
neurofibromatosis were donated for research. The donation of
abnormal PGD embryos for the derivation of hESCs was approved by
the ethical committee at the Hadassah Medical Center as well as the
Israeli Ministry of Health National Helsinki Committee for Genetic
Research in Humans. The embryos were cultured to the blastocyst
stage and the ICMs were isolated with the assistance of laser as
previously described [Turetsky T, Aizenman E, Gil Y, Weinberg N,
Shufaro Y, Revel A, Laufer N, Simon A, Abeliovich D, Reubinoff B E.
Laser-assisted derivation of human embryonic stem cell lines from
IVF embryos after preimplantation genetic diagnosis. Hum Reprod.
2008 January; 23(1):46-53]. Five isolated ICMs were transferred and
further cultured in the suspension culture system. The cells of one
of the ICMs proliferated leading to an increase in the size of the
cluster and generating new clusters. The clusters of cells were
passaged and expanded 6 weeks by mechanical dissection. They had
morphological characteristics similar to those of hESCs when
cultured in clusters under the same suspension culture conditions
being compacted clusters of uniformly small packed relatively
transparent cells which do not form cystic structures.
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